Fluorescence Measurement of Intracellular Sodium Concentration in Single Escherichia coli Cells  Chien-Jung Lo, Mark C. Leake, Richard M. Berry  Biophysical Journal  Volume 90, Issue 1, Pages 357-365 (January 2006) DOI: 10.1529/biophysj.105.071332 Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 1 (A) Typical bright-field image of a YS34 E. coli cell. (B) Fluorescence image of the same cell. Laser power 7.35 W/cm2, exposure time 1s. (C) The fluorescence image is divided into three regions: white, background region; red, marginal area; yellow, cell area. (D) Pixel intensity histogram illustrating the method of determining the different image regions used to obtain the fluorescence signal. See text for details. (E) An image intensity profile along the x axis in B (average of five pixel lines). The shaded area contributed to the fluorescence intensity signal. Biophysical Journal 2006 90, 357-365DOI: (10.1529/biophysj.105.071332) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 2 Fluorescence intensity of cells expressing chimeric motor proteins versus time of loading with 40μM Sodium Green in motility buffer. The measurements were made immediately after loading; 30min was chosen as the optimal loading time. Mean±SD of 10 cells is shown. Biophysical Journal 2006 90, 357-365DOI: (10.1529/biophysj.105.071332) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 3 Photobleaching of Sodium Green. (A) Fluorescence intensity of a single loaded cell as a function of time during continuous illumination at an intensity of 9.8 W/cm2, exposure time 1s. The curve is fitted as exponential decay with a time constant of 30±0.75s. (B) Photobleaching decay rates as a function of illumination intensity (mean±SD of five cells at each intensity). The rates are proportional to intensity as expected for photobleaching (shaded line). (C) A combined decay curve for data at different intensities. Photobleaching can be described as F(x) = Fo exp(−x/xo), where x is the accumulated laser exposure. Biophysical Journal 2006 90, 357-365DOI: (10.1529/biophysj.105.071332) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 4 Calibration method. (A) [Na+]ex was varied between 0 and 85mM with gramicidin and CCCP present to equilibrate the sodium concentrations across membrane. (B) Fluorescence intensity in response to changes of [Na+]ex; ∼3min was required for equilibration of [Na+]in. (C) Steady-state fluorescence intensity as a function of sodium concentration, with a fit to Eq. 1. Mean±SD of three successive measurements of F are shown. Biophysical Journal 2006 90, 357-365DOI: (10.1529/biophysj.105.071332) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 5 In vivo [Na+]in measurements. (A) The increase of [Na+]in in response to a step change of [Na+]ex from 1 to 85mM. The response for each cell was fitted as [Na+]in= A0+ A1 (1-exp{−t/t0}) and relative [Na+]in was defined as ([Na+]in-A0)/A1. Mean±SD of five cells are shown, and an exponential fit with t0=29±9s. (B) Successive [Na+]in measurements on a single cell expressing chimeric flagellar motors in different [Na+]ex as indicated in the upper column (85mM, ♦; 10mM, ■; 5mM, (; 1mM, (). Solid and dashed lines connect repeated measurements at the same [Na+]ex to guide the eye. (C) [Na+]in versus [Na+]ex for another cell of the same type. Each point is an average of three successive measurements, taken at 20s intervals 5min after solution exchange at each [Na+]ex. Error bars indicate the combined error, as described in the text. The shaded line is the power law fit, [Na+]in=A ([Na+]ex)α, with concentrations in mM, A=7.2±0.7, and α=0.20±0.03. (D) The same data as in C plotted as ΔpNa versus [Na+]ex. Error bars are converted from C, assuming no error in [Na+]ex. Biophysical Journal 2006 90, 357-365DOI: (10.1529/biophysj.105.071332) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 6 (A) [Na+]in versus [Na+]ex for eight individual cells expressing the chimeric flagellar motor. (B) The same data as in A plotted as ΔpNa versus [Na+]ex. (C) Scatter plots of fitted parameters from Eq. 1, Kd (left) and Fmax/Fmin (right), versus [Na+]in at [Na+]ex=85mM, for the same eight cells. There were no strong correlations between calibration parameters and [Na+]in. The standard deviations of Kd, Fmax/Fmin, and [Na+]in were 9.1%, 9.9%, and 29.9% of the mean values, respectively. Error bars in A and B are as in Fig. 5, C and D, respectively. Biophysical Journal 2006 90, 357-365DOI: (10.1529/biophysj.105.071332) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 7 (A) [Na+]in versus [Na+]ex in cells expressing chimeric PomA/PotB7E stators (□, eight cells) in cells containing the chimeric stator plasmid but grown without induced expression (, five cells) and in cells expressing wild-type MotA/MotB stators (▵, five cells). (B) The same data as in A plotted as ΔpNa versus [Na+]ex. Error bars indicate standard errors of the mean for each cell type and [Na+]ex. Biophysical Journal 2006 90, 357-365DOI: (10.1529/biophysj.105.071332) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 8 (A) Speed measurements in different [Na+]ex. Motor speeds were measured by back-focal-plane interferometry using 0.97μm diameter beads attached to truncated flagella. Thirty cells were measured at each [Na+]ex, under conditions where the number of stators could be determined by recording transient speed changes corresponding to addition or removal of individual stators. Mean±SD of 30 cells are shown. (B) Motor speed versus ΔpNa at the same [Na+]ex, determined as described in the text. The linear fit extrapolates to zero speed at a ΔpNa corresponding to +137mV. Biophysical Journal 2006 90, 357-365DOI: (10.1529/biophysj.105.071332) Copyright © 2006 The Biophysical Society Terms and Conditions